Accelerated Stability During Formulation Development of Early Stage Protein Therapeutics Pros and Cons of Contrasting Approaches

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1 Accelerated Stability During Formulation Development of Early Stage Protein Therapeutics Pros and Cons of Contrasting Approaches 2008 IBC Formulation Strategies for Protein Therapeutics Tim Kelly, Ph.D. Vice President, Biopharmaceutical Development KBI Biopharma, Inc.

2 Overview Description of Conservative and Aggressive approaches Tool Box Methodology Case Study: Aggressive Approach Case Study: Conservative Approach Pros and Cons of each Approach Conclusions

3 Stability Studies During Formulation Development The goal of formulation development is to determine a composition for the final dosage form that results in a safe, efficacious product which remains stable over the course of its intended use. Stability of various candidate formulations is generally evaluated during development via a combination of real-time and accelerated stability studies. Among drug companies, there is considerable divergence in the extent of stability data deemed appropriate to support formulation development during preclinical and phase I clinical development.

4 Conservative Approach A conservative approach for evaluating formulation stability may involve placing 10 or more candidate formulations in a long term stability study under realtime (e.g., 5 C) and accelerated (e.g., 30 C/65% RH) conditions. May often include platform formulations for a given product type The duration for such a study may range from 6 months to two years, with samples from each of the candidate formulations analyzed in one to three month intervals over the study duration.

5 Conservative Approach At the conclusion of the study, the candidate formulation that exhibits the best overall conservation of native purity and activity is chosen and utilized for manufacturing of clinical trial material. This conservative approach is most commonly utilized by large pharma companies with large product development pipelines who have the time and resources to permit this approach.

6 Conservative Approach Advantages Formulation decision is based on real-time stability data at the intended product storage temperature.» High degree of confidence that the product will remain stable throughout the duration of its intended use in clinical studies.» Nothing substitutes for real-time stability Disadvantages Time Consuming Labor Intensive Requires Large Quantity of API

7 Aggressive Approach Smaller pharma and biotech companies, especially those who do not yet have revenue streams from commercial products, often can t afford to devote the time and resources required to execute a 1-2 year real time stability study during formulation development. Such companies may take a more aggressive approach, with a greater emphasis on accelerated stability over a shorter duration. KBI Biopharma frequently employs an approach where a large number (30-40) of candidate formulations are evaluated via statistical design of experiments (DOE) including short term stability under real-time and accelerated conditions for months.

8 Aggressive Approach The accelerated stability conditions used in such studies may range from C to increase the rates of degradation and enhance the likelihood of observing significant differences among the candidate formulations over the short duration of the study. Understand thermal properties (e,g., Tm s of API) to set the stress temperature A long term stability study may then be performed on the selected formulation and ultimately on the actual clinical trial material prepared in the final selected formulation, in accordance with the ICH guidelines.

9 Aggressive Approach Advantages Acceleration of development timelines More efficient use of resources (people, lab, and API) Disadvantages Challenge of assigning predictive value to accelerated stability data for protein therapeutics.» Degradation processes that occur at accelerated temperatures may be irrelevant to shelf life The translation of accelerated stability data into a real-time storage shelf life is problematic as protein degradation processes are quite complex and often do not follow Arrhenius kinetics.» Can not be used to directly predict real time shelf life

10 Tool Box Methodology Our study is only as good as our methods Biophysical, Analytical, Biological Interrogate thermal, conformational, chemical, and biological stability Orthogonal Techniques e.g, SEC-HPLC and IEX-HPLC; DSC and DLS Conditions optimal for reducing formation of one impurity may be sub-optimal for others Designed to detect/quantify product-related impurities of interest HMW species, deamidation, oxidation, other charge variants, clips / truncated species Rugged with acceptable accuracy and precision, and understood variability Differentiate analytical variability from changes to product Suitable for use in statistical DOE

11 Case Study Aggressive Approach Monoclonal Antibody for IV administration ~25mg/mL Goal to initiate Phase I clinical trials as rapidly as possible Goal to develop IV formulation within 6 months Preformulation Development Forced Degradation Studies Final Dosage Form Development Utilized aggressive approach to identify lead candidate formulation based on statistical DOE, forced degradation and accelerated stability

12 Case Study Aggressive Approach Initial Linear Ranging Studies Solubility Biophysical characterization Forced Degradation Studies Freeze/Thaw Agitation Preformulation DOE Two 2-level factorial designs Two weeks accelerated stability Select Formulation Identify critical factors, Eliminate non-critical factors 6 Month Non-GMP Stability Study on Selected Formulation Stability on cgmp Drug Substance & Drug Product

13 Case Study Aggressive Approach Solubility Evaluated 5 buffer types, two ph values each» Acetate, Citrate, Histidine, Succinate, Phosphate Selected 2 buffer types for further evaluation, based on observed solubility and ease of concentration Forced Degradation Freeze-thaw and Agitation Evaluated excipients in each buffer system» Salt, Sucrose, Sorbitol, Arginine, Histidine, Polysorbate 20 Utilized Biophysical and Analytical techniques» DSC, CD, FTIR, SEC-HPLC, CEX-HPLC, SDS-PAGE Selected 2 excipient types for further evaluation

14 Case Study Aggressive Approach Accelerated Stability Two 2-level factorial designs 5 C (non-stressed) and 40 C/75%RH (stressed) for 2 weeks DSC, FTIR, SEC-HPLC, CEX-HPLC, SDS-PAGE, Bioassay Designed to optimize ph, buffer concentration, and excipient concentration for each candidate buffer / excipient system Evaluate interactions between formulation factors Interrogate thermal, conformational, chemical, and biological stability using a combination of biophysical and analytical techniques Understand and optimize the API design space DOE allows evaluation of statistical significance of differences observed between candidate formulations

15 Use of DOE in Accelerated Stability DOE Summary Two factorial designs Selected to span a wide range of ph conditions Full panel of analytics performed: SEC showed effect of buffer/ph on HMW species Phosphate buffer: HMW increases with increasing ph Histidine buffer: HMW stable from ph 6-7 Design-Expert Software HMW species Design Points D1 Histidine D2 Phosphate X1 = A: ph X2 = D: Buffer Type Actual Factors B: Buffer Conc = C: NaCl Conc = HMW species Interaction D: Buffer Type A: ph

16 Case Study Aggressive Approach Chose formulation and initiated a longer term (6 month) real time and accelerated stability study on the selected formulation Performed concurrently with process development, scale up, and Phase I bulk drug substance manufacturing Enabled and accelerated the transition to Phase I clinical manufacturing Mitigated risk of short-term stability study via follow-on 6 month study» Placed only the selected formulation up on stability for 6 months» Extensive package of storage conditions» -80 C, -20 C, 5 C Upright & Inverted, 30 C/60%RH Upright & Inverted

17 Case Study Aggressive Approach SEC Main Peak 99.00% Peak Area 98.00% 97.00% 96.00% 95.00% 94.00% 93.00% C -20C 5C upright 5C inverted 30C upright 30C inverted Time (months) SEC HMW Species Peak Area (%) 2.20% 2.10% 2.00% 1.90% 1.80% 1.70% 1.60% 1.50% Time (months) -80C -20C 5C upright 5C inverted 30C upright 30C inverted

18 Case Study Aggressive Approach CEX Main Peak Peak Area 65.00% 60.00% 55.00% 50.00% 45.00% 40.00% 35.00% 30.00% Time (months) -80C -20C 5C upright 5C Inverted 30C upright 30C Inverted CEX Acidic Species CEX Basic Species Peak Area 70.00% 60.00% 50.00% 40.00% 30.00% 20.00% 10.00% 0.00% Time (months) -80C -20C 5C upright 5C Inverted 30C upright 30C Inverted Peak Area 14.00% 12.00% 10.00% 8.00% 6.00% 4.00% Time (months) -80C -20C 5C upright 5C Inverted 30C upright 30C Inverted

19 Case Study Aggressive Approach Lane Sample Mark 12 MWM 100 ngbsa Intensity Marker 50 ng BSA Intensity Marker 5ºC, upright, 20 µg load 5ºC, upright, 5 µg load 5ºC, inverted, 20 µg load 5ºC, inverted, 5 µg load 30ºC, upright, 20 µg load 30ºC, upright, 5 µg load blank Reduced SDS-PAGE 6 months

20 Case Study Aggressive Approach Lane Sample 100 ngbsa Intensity Marker Mark 12 MWM 50 ng BSA Intensity Marker 5ºC, upright, 20 µg load 5ºC, upright, 5 µg load 5ºC, inverted, 20 µg load 5ºC, inverted, 5 µg load 30ºC, upright, 20 µg load 30ºC, upright, 5 µg load blank Non-reduced SDS-PAGE 6 months

21 Case Study Aggressive Approach cgmp Phase I Clinical Drug Product 6 Month Stability >98% Main Peak <2% HMW Species >63% Main Peak <28% Acidic Species <10% Basic Species

22 Case Study Conservative Approach Monoclonal Antibody for IV administration ~25mg/mL Goal to initiate Phase I clinical trials within 2-3 years Utilized a conservative approach with extensive real time and accelerated stability Nine candidate formulations placed on stability for 6 months Platform formulation, primarily one buffer system; goal to get some granularity within the buffer system (ph, excipients)» Four temperature conditions between 5 C and 45 C Three top candidates selected and placed on stability for months» Four temperature conditions between 5 C and 37 C SEC-HPLC, CEX-HPLC, SDS-PAGE, DLS

23 Case Study Conservative Approach SEC Main Peak, 5C Peak Area 98.00% 97.50% 97.00% 96.50% 96.00% Time (months) Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9 SEC Main Peak, 25C SEC Main Peak, 37C Peak Area 97.00% 96.50% 96.00% 95.50% 95.00% 94.50% 94.00% Time (months) Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9 Peak Area 96.00% 95.00% 94.00% 93.00% 92.00% 91.00% Time (months) Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9

24 Case Study Conservative Approach CEX Main Peak, 5C Peak Area 58.00% 57.50% 57.00% 56.50% 56.00% 55.50% 55.00% 54.50% 54.00% Time (months) Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9 CEX Main Peak, 25C CEX Main Peak, 37C Peak Area 65.00% 60.00% 55.00% 50.00% 45.00% 40.00% Time (months) Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9 Peak Area 45.00% 40.00% 35.00% 30.00% 25.00% Time (months) Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9

25 Case Study Conservative Approach CEX Acidic Species, 5C Peak Area 30.00% 28.00% 26.00% 24.00% 22.00% 20.00% Time (months) Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9 CEX Acidic Species, 25C CEX Acidic Species, 37C Peak Area 40.00% 35.00% 30.00% 25.00% 20.00% Time (months) Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9 Peak Area 60.00% 55.00% 50.00% 45.00% 40.00% 35.00% 30.00% 25.00% Time (months) Formulation 1 Formulation 2 Formulation 3 Formulation 4 Formulation 5 Formulation 6 Formulation 7 Formulation 8 Formulation 9

26 Case Study Conservative Approach Lane 1 Mark 12 Molecular Weight Standard Lane 2 Control Lane 3 25ºC, Formulation 9 Lane 4 5ºC, Formulation 9 Lane 5 25ºC, Formulation 8 Lane 6 5ºC, Formulation 8 Lane 7 25ºC, Formulation 7 Lane 8 5ºC, Formulation 7 Lane 9 25ºC, Formulation 6 Lane 10 5ºC, Formulation 6 Non-reduced SDS-PAGE, 6 Months

27 Case Study Conservative Approach Upon completion of 6 month study, three formulations selected and placed on stability for 12 months SEC HMW Species, 5C Peak Area 1.70% 1.50% 1.30% 1.10% 0.90% 0.70% 0.50% Time (months) Formulation 1 Formulation 2 Formulation 3 SEC HMW Species, 25C 2.00% Peak Area 1.50% 1.00% Formulation 1 Formulation 2 Formulation % Time (months)

28 Case Study Conservative Approach CEX Main Species, 5C CEX Main Species, 25C Peak Area 54.00% 52.00% 50.00% 48.00% 46.00% 44.00% 42.00% 40.00% Formulation 1 Formulation 2 Formulation 3 Peak Area 54.00% 52.00% 50.00% 48.00% 46.00% 44.00% 42.00% 40.00% Formulation 1 Formulation 2 Formulation Time (months) Time (months) CEX Acidic Species, 5C CEX Acidic Species, 25C 27.00% 40.00% Peak Area 25.00% 23.00% 21.00% 19.00% 17.00% 15.00% Formulation 1 Formulation 2 Formulation 3 Peak Area 35.00% 30.00% 25.00% 20.00% Formulation 1 Formulation 2 Formulation 3 Time (months) Time (months)

29 Aggressive vs. Conservative Aggressive - Pros Delivered formulation composition in approximately 4 months» Facilitated the completion of process development and scaleup activities» Completed 6 months of real-time non-gmp stability within 11 months of project initiation» Plenty of real-time stability data to support IND Resulting formulation showed excellent 5 C stability» 0.2% decrease in purity via SEC-HPLC after 6 months» No significant changes to CEX-HPLC or SDS-PAGE profile after 6 months» Similar profile for cgmp drug product lot after 6 months storage

30 Aggressive vs. Conservative Aggressive - Cons No 5 C real-time stability data beyond 6 months Virtually no real-time stability data on back-up formulations in the event of precipitous drop in purity after 6 months Statistical DOE and short term accelerated stability (2-4 weeks) may be used to identify formulations with acceptable long term real-time stability profiles Evaluate interactions between formulation factors to optimize design space Use biophysical characterization techniques to assess thermal and conformational stability of candidate formulations

31 Aggressive vs. Conservative Conservative - Pros Resulting formulations showed excellent 5 C stability» 0.5% increase in HMW species via SEC-HPLC after 12 months for all three formulations» 4% decrease in MP purity via CEX-HPLC after 12 months for all three formulations High level of confidence in ultimate shelf life of clinical supplies Strong fall back position of alternative formulations with extensive real-time stability to support their use should problems arise during manufacturing or early clinical development Plenty of real-time stability to support IND

32 Aggressive vs. Conservative Conservative Cons Observed very similar profiles for most formulations» 0.5% difference in HMW species via SEC-HPLC after 12 months for all three formulations» 4% difference in MP purity via CEX-HPLC after 12 months for all three formulations Value relative to resources expended and time spent Little/no evaluation of different buffer/ph systems» Platform approach, did not screen a broad range of buffers / ph values

33 Aggressive Approach

34 Conservative Approach

35 Conclusions MAb formulations with acceptable long term stability profiles may be developed via both aggressive and conservative approaches While the aggressive approach includes some elements of risk, it offers substantial potential benefits with respect to timelines and resources Statistical DOE and short term accelerated stability (2-4 weeks) may be used to identify formulations with acceptable long term real-time stability profiles The conservative approach offers significant risk mitigation via back up formulations A comprehensive toolbox of orthogonal analytical and biophysical techniques is essential in both cases, but particularly for the aggressive approach

36 Acknowledgements Vickie Dowling, PhD, Group Leader, Biopharmaceutical Development Khurshid Iqbal, PhD, Senior VP & CSO

37 Use of DOE in Accelerated Stability Des ign-expert Software PDI Design Points C1 200mM Sorbitol C2 125mM NaCl X1 = A: ph X2 = C: Excipient Interaction C: Excipient Actual Factor B: Buffer = Acetate PDI A: ph Linear response surface design Polydispersity index (PDI) via DLS Interaction between ph and excipient type

38 Case Study Aggressive Approach

39 Case Study Conservative Approach

40 Case Study Conservative Approach

41 Case Study Conservative Approach

42 Case Study Conservative Approach